Smooth muscle composes the walls of the blood vessels, the gastro- intestinal tract, and the urinary and reproductive system, and is absolutely essential for the proper function of these tissues. Calcium is the principal second messenger by which external stimuli regulate contraction in smooth muscle. Thus, in order to understand how contraction is controlled, it is essential to understand how the [Ca2+] is regulated. The goal of this project is to characterize, in single living cells, the mechanisms that are important for the movement of calcium into and out of the smooth muscle cytosol under resting conditions and in response to contractile stimuli. With the exception of the voltage-dependent calcium current, it has not been possible to measure, in single cells, the calcium flux of specific pumps and channels that regulate the [Ca2+] in response to stimuli. Although the development of [Ca2+] sensitive dyes has allowed routine measurements of free ionic [Ca2+] changes, cytosolic binding sites bind vastly more calcium than is present in the free ionic form. Thus, in the absence of accurate estimates of these calcium bind sites, these dyes do not reveal the magnitude or the time course of the underlying calcium fluxes. In this study, the voltage-clamp technique will be used in conjunction with high time resolution measurements of cytosolic [Ca2+] to study the regulation of [Ca2+] in single smooth muscle cell isolated from gastric and vascular tissue. The most important aim of this project will be to characterize the buffering ability of the cytosolic calcium binding sites. This information will allow the net calcium flux to be calculated directly from measurements of the rate of change of the free ionic [Ca2+]. By monitoring the rate of [Ca2+] decline after a period of elevation, calculations of the calcium efflux through the calcium pumps can be made from the rate of change of the free ionic [Ca2+]. In addition to allowing an assessment of the characteristics of the pumps that are responsible for the decline in [Ca2+], this efflux information will also provide a measure of the calcium efflux that is occurring whenever the [Ca2+] is above resting levels. Together, the ability to calculate the net flux and the efflux of calcium will allow the estimation of calcium release from internal stores. Thus, in situ measurements of specific calcium fluxes will be possible. By exploiting pharmacological or physical differences, further experiments will be conducted to characterize the specific calcium pumps responsible for lowering the [Ca2+] and the specific intracellular calcium pools that contribute to the rise in [Ca2+] when a cell is stimulated by various agents. The information obtained in this study should greatly increase our understanding of the regulation of contraction in smooth muscle and thus will ultimately help in the rational design of strategies to treat hypertension and other diseases that result from smooth muscle dysfunction.